This chapter focuses on the sequence-specific methylation of internal adenosine residues to form N6-methyladenosine. It talks about nucleosides modifications present in cellular mRNA; the nucleoside modifications present in virion RNA and viral mRNA are also be discussed because of the nearly complete overlap of the spectrum of modifications present. Lower eukaryotes such as yeast and Neurospora contain only cap 0. Collectively, these cap methylation events account for the presence of most of the nucleoside modifications in eukaryotic mRNA, namely, 7-methylguanine (m7G), all four 2’-O-methylnucleosides (Nm), and N6,2’-Odimethyl-adenosine (m6Am). Three modified nucleosides have been reported within internal regions of eukaryotic mRNA: N6methyladenosine (m6A), 5-methylcytidine (m5C), and inosine. A study examined short (20 nucleotide) synthetic RNA substrates and confirmed that the in vitro specificity closely paralleled the frequency with which sequences are methylated in vivo as originally described by Schibler. This study was extended by evaluating an additional two m6A sites in a similar fashion. Camper and his coworkers utilized an inhibitor, S-tubericidinylhomocysteine (STH), which is a structural analog of S-adenosylmethionine (SAM) and a potent inhibitor of SAM-dependent methyltransferases. In this study, the effects of the inhibitor on cap methylation and internal methylation were closely monitored. Posttranscriptional modification of nucleosides within mRNA molecules is an integral aspect of premRNA processing. These modifications are characteristic of eukaryotic mRNA only; no equivalent nucleotide modifications have been found in any of the prokaryotic mRNAs characterized so far.

Chemical structure of eukaryotic mRNA cap. The general structure of a typical mRNA cap is shown. The terminal N7-methylguanosine is present linked to the first transcribed nucleoside (N1) via a 5′-5′-triphosphate bridge. If neither N1 nor N2 is methylated at the 2′ - O position, the structure is designated cap 0. If N1 is methylated at the 2′ - O position, the structure is designated cap 1. If N1 and N2 are methylated at the 2′ - O position, the structure is designated cap 2.

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Figure 1

Chemical structure of eukaryotic mRNA cap. The general structure of a typical mRNA cap is shown. The terminal N7-methylguanosine is present linked to the first transcribed nucleoside (N1) via a 5′-5′-triphosphate bridge. If neither N1 nor N2 is methylated at the 2′ - O position, the structure is designated cap 0. If N1 is methylated at the 2′ - O position, the structure is designated cap 1. If N1 and N2 are methylated at the 2′ - O position, the structure is designated cap 2.

Potential secondary structure of mRNAs containing m6A sites. Partial sequences of mRNA from bPRL and RSV surrounding selected m6A sites were analyzed for potential secondary structures using the method of Zuker (1989). The methylation consensus sequences are highlighted in gray; the methylated adenosyl residue is shown in larger type. (A) 62 nucleotides of sequence corresponding to the 3′-terminus of the bPRL mRNA. This is also the sequence of the RNA used in in vitro assays for the purification of m6A-MT (Bokar et al. 1994, 1997). (B) 105 nucleotides of sequence corresponding to the first methylation site in the RSV src gene at position 7414 (Kane and Beemon, 1985). (C) 42 nucleotides of sequence corresponding the methylation site at position 6718 in the RSV env gene (Kane and Beemon, 1985).

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Figure 2

Potential secondary structure of mRNAs containing m6A sites. Partial sequences of mRNA from bPRL and RSV surrounding selected m6A sites were analyzed for potential secondary structures using the method of Zuker (1989). The methylation consensus sequences are highlighted in gray; the methylated adenosyl residue is shown in larger type. (A) 62 nucleotides of sequence corresponding to the 3′-terminus of the bPRL mRNA. This is also the sequence of the RNA used in in vitro assays for the purification of m6A-MT (Bokar et al. 1994, 1997). (B) 105 nucleotides of sequence corresponding to the first methylation site in the RSV src gene at position 7414 (Kane and Beemon, 1985). (C) 42 nucleotides of sequence corresponding the methylation site at position 6718 in the RSV env gene (Kane and Beemon, 1985).

Effects of methylation inhibitors on the cytoplasmic appearance of newly synthesized RNA and on nuclear splicing of a bPRL derived pre-mRNA. (A) Effect of a methylation inhibitor on the cytoplasmic appearance of newly synthesized RNA. HeLa cells were prelabeled with a low level of (14C]uridine (0.48 mCi/mmol) for 12 h, treated with 500 μΜ STH (open circles) or no STH (closed circles) for 90 min, and then labeled with [3H]uridine (40 mCi/mmol). At various times after the addition of [3H]uridine, cells were placed on ice, and cytoplasmic poly(A)+ RNA and nonpolyadenylated RNA were prepared subsequently. The ratio of [3H]uridine to (14C]uridine in cytoplasmic poly(A)+ RNA and nonpolyadenylated RNA is shown. Appearance of newly synthesized polyadenylated RNA was delayed in STH treated cells as compared to control cells. No difference was seen for nonpolyadenylated RNA (inset). (B) Quantitative S1 nuclease mapping of nuclear bPRL precursor and mature-form RNA in stably transfected cells treated with the methylation inhibitor neplanocin (NPC). An autoradiogram of a gel showing the DNA fragments protected by 5 µg of total nuclear RNA isolated from cells treated with 10 μΜ NPC for 8 h (+NPC) and untreated cells (control). The unspliced (precursor) and spliced (mature) bPRL minigene transcripts are shown. The lane labeled mock contains probe that was hybridized in the absence of RNA followed by treatment with S1 nuclease.

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Figure 3

Effects of methylation inhibitors on the cytoplasmic appearance of newly synthesized RNA and on nuclear splicing of a bPRL derived pre-mRNA. (A) Effect of a methylation inhibitor on the cytoplasmic appearance of newly synthesized RNA. HeLa cells were prelabeled with a low level of (14C]uridine (0.48 mCi/mmol) for 12 h, treated with 500 μΜ STH (open circles) or no STH (closed circles) for 90 min, and then labeled with [3H]uridine (40 mCi/mmol). At various times after the addition of [3H]uridine, cells were placed on ice, and cytoplasmic poly(A)+ RNA and nonpolyadenylated RNA were prepared subsequently. The ratio of [3H]uridine to (14C]uridine in cytoplasmic poly(A)+ RNA and nonpolyadenylated RNA is shown. Appearance of newly synthesized polyadenylated RNA was delayed in STH treated cells as compared to control cells. No difference was seen for nonpolyadenylated RNA (inset). (B) Quantitative S1 nuclease mapping of nuclear bPRL precursor and mature-form RNA in stably transfected cells treated with the methylation inhibitor neplanocin (NPC). An autoradiogram of a gel showing the DNA fragments protected by 5 µg of total nuclear RNA isolated from cells treated with 10 μΜ NPC for 8 h (+NPC) and untreated cells (control). The unspliced (precursor) and spliced (mature) bPRL minigene transcripts are shown. The lane labeled mock contains probe that was hybridized in the absence of RNA followed by treatment with S1 nuclease.

Purification of the MT-A70 subunit of mRNA m6A-MT. MT-A was purified as previously described (Bokar et al., 1994) by cation exchange, anion exchange, and size exclusion chromatography. The active fractions were pooled and further fractionated on a heparin-Sepharose column. (A) A representative activity profile of the heparin-Sepharose column fractions is shown. Closed circles represent the activity of aliquots of the fractions when assayed with supplemented partially purified fractions of MT-B; open circles are without MT-B. The KCl concentration units are molar. (B) [3H-methyl]SAM-UV-cross-linking. Aliquots of the same fractions assayed in panel A were cross-linked to [3H-methyl]SAM using UV light, and were then separated by SDS-8% PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and fluorographed. The 70-kDa band that coelutes with MT-A activity is shown (MT-A70). For details of experimental methodology see Bokar et al., 1994 and 1997.

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Figure 4

Purification of the MT-A70 subunit of mRNA m6A-MT. MT-A was purified as previously described (Bokar et al., 1994) by cation exchange, anion exchange, and size exclusion chromatography. The active fractions were pooled and further fractionated on a heparin-Sepharose column. (A) A representative activity profile of the heparin-Sepharose column fractions is shown. Closed circles represent the activity of aliquots of the fractions when assayed with supplemented partially purified fractions of MT-B; open circles are without MT-B. The KCl concentration units are molar. (B) [3H-methyl]SAM-UV-cross-linking. Aliquots of the same fractions assayed in panel A were cross-linked to [3H-methyl]SAM using UV light, and were then separated by SDS-8% PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and fluorographed. The 70-kDa band that coelutes with MT-A activity is shown (MT-A70). For details of experimental methodology see Bokar et al., 1994 and 1997.

An anti-MT-A70 immunoreactive protein copurifies with MT-A activity. (A) A representative activity profile from a Mono S (cation exchange) column is shown. Closed circles represent the activity of aliquots of the fractions when assayed with supplemented partially purified fractions of MT-B; open circles are without MT-B. The NaCl concentration units are molar. MT-A activity elutes mainly in fractions 14 and 15. (B) Western blot analysis of Mono S column fractions. Aliquots of the fractions were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with anti-MT-A70 antisera and then a horseradish peroxidase conjugated secondary antibody, and bands were visualized by chemiluminescence and fluorography. A 70-kDa immunoreactive protein coelutes with methyltransferase activity. (For details of experimental methodology, see Bokar et al., 1997.)

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Figure 5

An anti-MT-A70 immunoreactive protein copurifies with MT-A activity. (A) A representative activity profile from a Mono S (cation exchange) column is shown. Closed circles represent the activity of aliquots of the fractions when assayed with supplemented partially purified fractions of MT-B; open circles are without MT-B. The NaCl concentration units are molar. MT-A activity elutes mainly in fractions 14 and 15. (B) Western blot analysis of Mono S column fractions. Aliquots of the fractions were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with anti-MT-A70 antisera and then a horseradish peroxidase conjugated secondary antibody, and bands were visualized by chemiluminescence and fluorography. A 70-kDa immunoreactive protein coelutes with methyltransferase activity. (For details of experimental methodology, see Bokar et al., 1997.)

MT-A70 sequence homology to bacterial N6-adenine DNA methyltransferase motifs and to SP08. (A) Alignment of similar regions of MT-A70 and M. Mun1 methyltransferase. A vertical line denotes identical amino acids, and a colon denotes similar amino acids. The regions that correspond to consensus DNA methyltransferase motifs are highlighted (see Timinskas et al., 1995). (B) Alignment of similar regions of MT-A70 and SPOS. (C) Schematic representation of three groups of EST sequences that contain regions of identity to the 3′-portion pMT-A70. The stippled box represents pMT-A70 sequence. The thin lines represent EST sequence not homologous to pMT-A70. Blocks of sequence that appear to be alternatively included exons that can be deduced from alignment of the EST sequences are denoted by lowercase roman numerals. The GenBank accession numbers for the EST sequences are as follows: type I, N39589 and W93679; type II, N55548, W04670, R42072, and W95745; and type III, N94880, N95688, N66219, W58127, F09834, N66255, and C00061.

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Figure 6

MT-A70 sequence homology to bacterial N6-adenine DNA methyltransferase motifs and to SP08. (A) Alignment of similar regions of MT-A70 and M. Mun1 methyltransferase. A vertical line denotes identical amino acids, and a colon denotes similar amino acids. The regions that correspond to consensus DNA methyltransferase motifs are highlighted (see Timinskas et al., 1995). (B) Alignment of similar regions of MT-A70 and SPOS. (C) Schematic representation of three groups of EST sequences that contain regions of identity to the 3′-portion pMT-A70. The stippled box represents pMT-A70 sequence. The thin lines represent EST sequence not homologous to pMT-A70. Blocks of sequence that appear to be alternatively included exons that can be deduced from alignment of the EST sequences are denoted by lowercase roman numerals. The GenBank accession numbers for the EST sequences are as follows: type I, N39589 and W93679; type II, N55548, W04670, R42072, and W95745; and type III, N94880, N95688, N66219, W58127, F09834, N66255, and C00061.

Northern blot analysis of MT-A70 mRNA. 32P-labelled probe was prepared by random primer labelling of a pMT-A70 cDNA insert and was hybridized to blots containing 2 μg of human tissue poly(A)+ RNA. The blots were washed using high-stringency conditions and exposed to X-ray film overnight with an intensifying screen. Two RNA species of approximately 2 and 3 kb were seen in each tissue sample. The blots were stripped and rehybridized with a probe specific for β-actin mRNA.

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Figure 7

Northern blot analysis of MT-A70 mRNA. 32P-labelled probe was prepared by random primer labelling of a pMT-A70 cDNA insert and was hybridized to blots containing 2 μg of human tissue poly(A)+ RNA. The blots were washed using high-stringency conditions and exposed to X-ray film overnight with an intensifying screen. Two RNA species of approximately 2 and 3 kb were seen in each tissue sample. The blots were stripped and rehybridized with a probe specific for β-actin mRNA.